Microorganisms useful in a method of producing α-halo-α, β-saturated carbonyl compounds

ABSTRACT

A method of producing an α-halo-α,β-saturated carbonyl compound from an α-halocarbonyl compound having an α,β-carbon-carbon double bond by reducing said α,β-carbon-carbon double bond using a microorganism belonging to any one of the genera Acetobacter, Actinomyces, Acinetobacter, Agrobacterium, Aeromonas, Alcaligenes, Arthrobacter, Azotobacter, Bacillus, Brevibacterium, Burkholderia, Cellulomonas, Corynebacterium, Enterobacter, Enterococcus, Escherichia, Flavobacterium, Gluconobacter, Halobacteium, Halococccus, Klebsiella, Lactobacillus, Microbacterium, Micrococcus, Micropolyspora, Mycobacterium, Nocardia, Pseudomonas, Pseudonocardia, Rhodococcus, Rhodobacter, Serratia, Staphylococcus, Streptococcus and Streptomyces, Xanthomonas, or a microbial product thereof. Pseudomonas sp. SD810, SD811 and SD812, Burkholderia sp. SD 816, and mutants thereof having an activity of reducing the α,β-carbon-carbon double bond of an α-halocarbonyl compound having an α,β-carbon-carbon double bond.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of application Ser. No. 09/369,144, now U.S. Pat.No. 6,379,935 filed Aug. 5, 1999, the disclosure of which isincorporated herein by reference. This application is an applicationfiled under 35 U.S.C. §111(a) claiming benefit pursuant to 35 U.S.C.§119(e)(i) of the filing date of the Provisional Application No.60/138,085 filed Jun. 8, 1999 pursuant to 35 U.S.C. §111(b).

FIELD OF THE INVENTION

The present invention relates to a method of producing a correspondingα-halo-α,β-saturated carbonyl compound from an α-halocarbonyl compoundhaving an α,β-carbon-carbon double bond by hydrogenating theα,β-carbon-carbon double bond using a microorganism belonging to thegenus Acetobacter, Actinomyces, Acinetobacter, Agrobacterium, Aeromonas,Alcaligenes, Arthrobacter, Azotobacter, Bacillus, Brevibacterium,Burkholderia, Cellulomonas, Corynebacterium, Enterobacter, Enterococcus,Escherichia, Flavobacterium, Gluconobacter, Halobacteium, Halococccus,Klebsiella, Lactobacillus, Microbacterium, Micrococcus, Micropolyspora,Mycobacterium, Nocardia, Pseudomonas, Pseudonocardia, Rhodococcus,Rhodobacter, Serratia, Staphylococcus, Streptococcus, Streptomyces orXanthomonas, preferably a microorganism belonging to the genusPseudomonas or Burkholderia, more preferably Pseudomonas sp. SD810,Pseudomonas sp. SD811, Pseudomonas sp. SD812 or Burkholderia sp. SD816,or a microbial product thereof. The present invention also relates tonovel microorganisms belonging to the genera Pseudomonas andBurkholderia, particularly Pseudomonas sp. SD810, Pseudomonas sp. SD811,Pseudomonas sp. SD812 and Burkholderia sp. SD816.

Furthermore, the present invention relates to a method of producing acorresponding α-halo-α,β-saturated carbonyl compound as an S formcompound with respect to the α-position from an α-halocarbonyl compoundhaving an α,β-carbon-carbon double bond by hydrogenating thecarbon—carbon double bond. This method can be used in the production ofoptically active carbonyl compounds such as various optically active(having an absolute S form configuration at the α-position) saturatedcarboxylic acids or amides. The optically active carbonyl compounds area highly valuable chiral building block which is difficult to prepare byclassical chemical processes, and are materials useful particularly as araw material of medical or agricultural chemicals.

BACKGROUND OF THE INVENTION

In recent years, a method of producing various compounds, particularlyoptically active substances, by the reduction of a carbon—carbon doublebond using a microorganism is drawing attention. To this effect, variousmethods of producing a corresponding α,β-saturated carbonyl compoundhaving a substituent at the α-position from a carbonyl compound havingan α,β-carbon-carbon double bond and having a substituent at theα-position by microbially reducing the carbon—carbon double bond havebeen reported (see, H. Simon, et al., Hoppe-Seyler's Z. Physiol. Chem.,362, 33 (1981), H. Giesel, et al., Arch. Microbiol., 135, 51 (1983), H.G. W. Leuenberger, et al., Helv. Chim. Acta., 62, 455 (1979), R.Matsuno, et al., J. Ferm. Bioeng., 84, 195 (1997)). However, forexample, according to the method of using bacteria as the microorganism,an anaerobe such as Clostridium kluyveri (DSM-555) or Clostridium sp.La-1 (DSM-1460) is used. Therefore, the growing rate of themicroorganism is slow, it is difficult to increase the cellconcentration and accordingly, the reaction rate is not satisfactorilyhigh. Thus, these methods have a problem in profitability andoperability.

The method using Clostridium theremosaccharolyticum disclosed inJP-A-63-003794 (the term “JP-A” as used herein means an “unexaminedpublished Japanese patent application”) has an object of solving theabove-descried problem by using a thermophilic bacterium. However, thebacterium used is still anaerobic, therefore, the growing rate and thereaction rate both are not satisfactorily high and the process involvesuse of hydrogen. Thus, the method fails in solving the problems inprofitability and safety. Furthermore, the α,β-saturated carbonylcompound having a substituent at the α-position produced by reducing aprochiral carbonyl compound having an α,β-carbon-carbon double bond andhaving a substituent at the α-position using a microorganism is acompound having an absolute R form configuration and an S formconfiguration compound cannot be produced.

The method of reducing the α,β-carbon-carbon double bond using a breadyeast as the microorganism has general-purpose applicability becausecompounds over a wide range can be reduced. In addition, since themicroorganism used is aerobic, good operability can be attained.Furthermore, the optically active substances produced include both Sform and R form, therefore, this method is most abundant in the casesreported. However, the yeast grows slowly as compared with bacteria, theoptical selectivity is not sufficiently high in many cases in thereduction reaction for obtaining a more optically active product, andreduction of an α-halocarbonyl compound having an α,β-carbon-carbondouble bond is not known.

As described above, in the technique of producing a correspondingα-halo-α,β-saturated carbonyl compound from an α-halocarbonyl compoundhaving an α,β-carbon-carbon double bond by reducing the carbon—carbondouble bond using a microorganism, a method satisfying all of therequirements regarding operability, profitability, safety and reactionproperties is not yet known.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method of producing acorresponding α-halo-α,β-saturated carbonyl compound from anα-halocarbonyl compound having an α,β-carbon-carbon double bond byreducing the carbon—carbon double bond using a microorganism, whichmethod can satisfy all of the requirements for operability,profitability, safety and reaction properties and ensure excellentoptical selectivity.

As a result of thorough screening from soil, the present inventors havefound that surprisingly, microorganisms capable of producing acorresponding α-halo-α,β-saturated carbonyl compound from anα-halocarbonyl compound having an α,β-carbon-carbon double bond byreducing the carbon—carbon double bond are distributed over a relativelywide genus range of the aerobes and facultative anaerobes. Inparticular, it has been found that strains having this activity arepresent in a large number in microorganisms belonging to the generaPseudomonas and Burkholderia, and some of these strains can reduce anα-halocarbonyl compound having an α,β-carbon-carbon double bond andthereby produce an extremely high-purity α-halo-α,β-saturated carbonylcompound having an absolute configuration of S form at the α-position.The present invention has been accomplished based on these findings.

More specifically, the present invention relates to the followingembodiments:

[1] a method of producing an α-halo-α,β-saturated carbonyl compound froman α-halocarbonyl compound having an α,β-carbon-carbon double bond byreducing the α,β-carbon-carbon double bond using a microorganismbelonging to any one of the genera Acetobacter, Actinomyces,Acinetobacter, Agrobacterium, Aeromonas, Alcaligenes, Arthrobacter,Azotobacter, Bacillus, Brevibacterium, Burkholderia, Cellulomonas,Corynebacterium, Enterobacter, Enterococcus, Escherichia,Flavobacterium, Gluconobacter, Halobacteium, Halococccus, Klebsiella,Lactobacillus, Microbacterium, Micrococcus, Micropolyspora,Mycobacterium, Nocardia, Pseudomonas, Pseudonocardia, Rhodococcus,Rhodobacter, Serratia, Staphylococcus, Streptococcus, Streptomyces andXanthomonas, or a microbial product thereof;

[2] the method of producing an α-halo-α,β-saturated carbonyl compound asdescribed in [1], wherein the α,β-carbon-carbon double bond of theα-halocarbonyl compound having an α,β-carbon-carbon double bond isreduced using a microorganism belonging to the genus Pseudomonas or amicrobial product thereof;

[3] the method of producing an α-halo-α,β-saturated carbonyl compound asdescribed in [1], wherein the α,β-carbon-carbon double bond of theα-halocarbonyl compound having an α,β-carbon-carbon double bond isreduced using a microorganism belonging to the genus Burkholderia or amicrobial product thereof;

[4] the method of producing an α-halo-α,β-saturated carbonyl compound asdescribed in [2], wherein the microorganism belonging to the genusPseudomonas is Pseudomonas sp. SD810;

[5] the method of producing an α-halo-α,β-saturated carbonyl compound asdescribed in [2], wherein the microorganism belonging to the genusPseudomonas is Pseudomonas sp. SD811;

[6] the method of producing an α-halo-α,β-saturated carbonyl compound asdescribed in [2], wherein the microorganism belonging to the genusPseudomonas is Pseudomonas sp. SD812;

[7] the method of producing an α-halo-α,β-saturated carbonyl compound asdescribed in [3], wherein the microorganism belonging to the genusBurkholderia is Burkholderia sp. SD816;

[8] the method of producing an α-halo-α,β-saturated carbonyl compound asdescribed in [1] to [7], wherein an S-form compound chiral at theα-position is produced by the reduction of the carbon—carbon doublebond;

[9] the method of producing an α-halo-α,β-saturated carbonyl compound asdescribed in [1] to [8], wherein the a-halocarbonyl compound having anα,β-carbon-carbon double bond is a compound represented by the followingformula (1):

wherein R₁ represents a halogen atom, R₂ and R₃ each independentlyrepresents a hydrogen atom, a halogen atom, a linear or branchedaliphatic hydrocarbon group having from 1 to 6 carbon atoms, a linear orbranched alkoxy group having from 1 to 6 carbon atoms, a hydroxyl group,a carboxyl group, an aromatic group which may be substituted, or anitrogen-, oxygen- or sulfur-containing heterocyclic group, and R₄represents a hydroxyl group, a linear or branched alkoxy group havingfrom 1 to 3 carbon atoms or a primary, secondary or tertiary aminogroup, and the α-halo-α,β-saturated carbonyl compound is a compoundrepresented by the following formula (2):

wherein R₁ to R₄ have the same meanings as defined above;

[10] the method of producing an α-halo-α,β-saturated carbonyl compoundas described in [9], wherein the compound represented by formula (1) isan α-haloacrylic acid and the compound represented by formula (2) is anα-halopropionic acid having an absolute S form configuration;

[11] the method of producing an α-halo-α,β-saturated carbonyl compoundas described in [10], wherein the halogen atom is a bromine atom;

[12] the method of producing an α-halo-α,β-saturated carbonyl compoundas described in [10], wherein the halogen atom is a chlorine atom;

[13] the method of producing an α-halo-α,β-saturated carbonyl compoundas described in [1] to [12], wherein the microbial product of amicroorganism is a microorganism culture, a microbial extract, amicrobial cell suspension or a microbial cell fixed to a support;

[14] the method of producing an α-halo-α,β-saturated carbonyl compoundas described in [1] to [13], wherein the microorganism used is variednot to decompose the α-halo-α,β-saturated carbonyl compound produced,thereby increasing the amount of the product accumulated;

[15] the method of producing an α-halo-α,β-saturated carbonyl compoundas described in [1] to [14], wherein the α-halocarbonyl compound havingan α,β-carbon-carbon double bond and a compound capable of beingoxidized by the microorganism used are present together in the reactionsystem and thereby the reaction continuing time is prolonged;

[16] the method of producing an α-halo-α,β-saturated carbonyl compoundas described in [15], wherein the compound capable of being oxidized bythe microorganism used is a sugar having from 3 to 6 carbon atoms;

[17] the method of producing an α-halo-α,β-saturated carbonyl compoundas described in [15], wherein the compound capable of being oxidized bythe microorganism used is an organic acid having from 2 to 4 carbonatoms;

[18] Pseudomonas sp. SD810 and mutants thereof having an activity ofreducing the α,β-carbon-carbon double bond of an α-halocarbonyl compoundhaving an α,β-carbon-carbon double bond;

[19] Pseudomonas sp. SD811 and mutants thereof having an activity ofreducing the α,β-carbon-carbon double bond of an α-halocarbonyl compoundhaving an α,β-carbon-carbon double bond;

[20] Pseudomonas sp. SD812 and mutants thereof having an activity ofreducing the α,β-carbon-carbon double bond of an α-halocarbonyl compoundhaving an α,β-carbon-carbon double bond;

[21] Burkholderia sp. SD816 and mutants thereof having an activity ofreducing the α,β-carbon-carbon double bond of an α-halocarbonyl compoundhaving an α,β-carbon-carbon double bond;

[22] a microbial product containing the microorganism described in [12]to [21]; and

[23] the microbial product as described in [22], which is a microbialculture, a microbial extract, a microbial cell suspension or a microbialcell fixed to a support.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an example of the accumulative production ofα-chloropropionic acid in the cases where a substance to be oxidized isnot present and where such is present.

FIG. 2 shows an example of the accumulative production ofα-chloropropionic acid in the cases where glucose is not present andwhere such is present.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The microorganisms which can be used in the present invention aremicroorganisms belonging to any one of the genera Acetobacter,Actinomyces, Acinetobacter, Agrobacterium, Aeromonas, Alcaligenes,Arthrobacter, Azotobacter, Bacillus, Brevibacterium, Burkholderia,Cellulomonas, Corynebacterium, Enterobacter, Enterococcus, Escherichia,Flavobacterium, Gluconobacter, Halobacteium, Halococccus, Klebsiella,Lactobacillus, Microbacterium, Micrococcus, Micropolyspora,Mycobacterium, Nocardia, Pseudomonas, Pseudonocardia, Rhodococcus,Rhodobacter, Serratia, Staphylococcus, Streptococcus, Streptomyces andXanthomonas.

Among these, microorganisms belonging to the genera Pseudomonas andBurkholderia are preferred. The strain is not particularly limited aslong as it has an activity of reducing the α,β-carbon-carbon double bondof an α-halocarbonyl compound having an α,β-carbon-carbon double bond.However, for example, Pseudomonas sp. SD810, Pseudomonas sp. SD811,Pseudomonas sp. SD812 and Burkholderia sp. SD816 are preferably used.Among these, Pseudomonas sp. SD811 and Burkholderia sp. SD816 are morepreferred. The strains Pseudomonas sp. SD810, Pseudomonas sp. SD811,Pseudomonas sp. SD812 and Burkholderia sp. SD816 are strains isolatedfrom soil and have an activity of decomposing and assimilating variouscarbonyl compounds.

The above-described microorganisms may be any of a wild type, variantand recombinant induced by cell fusion or genetic engineering as long asthe strain has an activity of reducing the carbon—carbon double bond ofan α-halocarbonyl compound having an α,β-carbon-carbon double bond. Forexample, a variant reduced or defective in activity of decomposing theproduct, a variant or recombinant improved in reducing activity or avariant improved in resistance against a high-concentration substrate orproduct may be preferably used.

Examples of the isolation and cultivation of these strains are describedbelow.

5 ml of a minimum medium obtained by adding, as a substantially solecarbon source, 2 g/l of an α,β-unsaturated carbonyl compound having ahalogen atom as a substituent at the α-position, such as α-chloroacrylicacid, to an inorganic salt culture medium (for example, (NH₄)₂SO₄: 2g/l, NaH₂PO₄: 1 g/l, K₂HPO₄: 1 g/l, MgSO₄: 0.1 g/l, yeast extract: 0.5g/l) used for normal bacteria is poured in a test tube and sterilized.Thereafter, about 0.1 g of soil is added thereto and cultivation byshaking is performed at 28° C. Subculture is performed every single day.After repeating this accumulative cultivation for 6 days, the culture isspread on the agar plate of minimum medium described above containing 20g/l of agar as a solidifying agent, and cultured at 28° C. for 3 days.By the isolation of colonies produced, the strain Pseudomonas sp. SD810,Pseudomonas sp. SD811 or Pseudomonas sp. SD812 may be obtained. Thesestrains Pseudomonas sp. SD810, Pseudomonas sp. SD811 and Pseudomonas sp.SD812 are deposited with National Institute of Bioscience andHuman-Technology at 1-3, Higashi 1 chome Tsukuba-shi Ibaraki-ken305-8566, JAPAN under the accession numbers Pseudomonas sp. SD810:BP-6767 (FERM BP-6767) (transferred from accession number 16746(FERM-16746)), Pseudomonas sp. SD811: BP-6768 (FERM BP-6768)(transferred from accession number 16747 (FERM-16747)) and Pseudomonassp. SD812: BP-6769 (FERM BP-6769) (transferred from accession number16748 (FERM-16748)), respectively. All deposited specimens described inthis application were deposited on Jun. 28, 1999.

Examples of the isolation and cultivation of the strains having arelatively small population in isolation source are described below.

By accumulative acclimatization using soil as the separation source,microorganisms having a small population, microorganisms having a lowassimilating activity or microorganisms having a low resistance againstα,β-unsaturated carbonyl compounds having a halogen atom as thesubstituent at the α-position (hereinafter sometimes referred to as a“halogen-containing compound”), such as α-chloroacrylic acid, may beseparated. More specifically, 0.01% (in the present specification,unless otherwise indicated, “%” is “weight (g)/volume (100 ml)×100”) ofvarious halogen-containing compounds are added as a substantially solecarbon source or added together with glucose in the same concentrationto a basal medium containing yeast extract (0.5 g/l), ammonium sulfate(2 g/l), sodium dihydrogenphosphate (1 g/l), dipotassiumhyrogenphosphate (1 g/l) and magnesium sulfate (0.1 g/l) (for theisolation of some microorganisms which require a high salt concentrationto grow, a medium obtained by adding sodium chloride in a concentrationof from 10 to 20% to the above-descried basal medium is used as thebasal medium). In 10 ml of the thus-prepared medium, 1 g of soilcollected is suspended and cultured by shaking at 30° C. After 24 hours,5 ml of the supernatant of the culture is sampled and after addingthereto 5 ml of a new medium having the same composition as above exceptthat the concentration of halogen-containing compounds is 0.02%,cultured by shaking at 30° C. (first subculture). After 24 hours, 5 mlof the supernatant of the culture was sampled and after adding thereto 5ml of the medium where the concentration of halogen-containing compoundsin the medium is increased from 0.02% to 0.1%, cultured by shaking at30° C. (second subculture). This operation is further repeated 3 timesevery 24 hours (third to fifth subculture). At the sixth subculture, theconcentration of halogen-containing compounds in the medium added to theculture is increased to 0.2% and 5 ml of the supernatant is sampled andafter adding thereto 5 ml of a new medium, cultured by shaking at 30° C.This operation is repeated 6 times (sixth to twelfth subculture) every24 hours. At each time of these 6 subculture operations, a part of thesupernatant of the culture is spread on a agar plate obtained by adding2% of agar to the above-described medium containing 0.2% of halogencontaining compounds, and cultured at 30° C. The colonies produced arepure separated and then, for example, Burkholderia sp. SD816 may beobtained. Burkholderia sp. SD816 is deposited with National Institute ofBioscience and Human-Technology under the accession number BP-6770 (FERMBP-6770).

The taxonomic test results of these strains are shown below.

Pseudomonas sp. SD810 Morphology: (1) Shape and size of cell rod 0.6-1.0μm × 1.2-3.0 μm (2) Motility motile (3) Gram staining negative (4) Sporenone (5) Bacteriolysis by 3% KOH positive Physiological activity: (1)Aminopeptidase positive (2) Oxidase positive (3) Catalase positive (4)Production of indole negative (5) VP test negative (6) Reduction ofnitrate negative (7) Denitrification negative (8) Use of citric acid(Simons) positive (9) Urease negative (10) Phenylalanine deaminasenegative (11) Use of maronic acid positive (12) Production of levan fromsucrose positive (13) Lecithinase negative (14) Hydrolysis of starchnegative (15) Hydrolysis of gelatin negative (16) Hydrolysis of caseinnegative (17) Hydrolysis of DNA negative (18) Hydrolysis of Tween 80negative (19) Hydrolysis of exrin negative (20) Growth Behavior tooxygen obligately aerobic Growth at 37° C. − Growth at 41° C. − Growthat pH 5.6 + Growth in Mac-Conkey-Agar medium − Growth in 55-Agar medium− Growth in Cetrimid-Agar − (21) Production of dye − Nondiffusivenegative Diffusive negative Fluorescent negative Pyrocyanine negative(22) OF Test no decomposition of sugar (23) Formation of acids Glucosenegative Fructose positive Xylose negative (24) ONPG (β-Galactosidase)negative (25) Arginine dihydrolase negative (26) Production of gas fromglucose negative (27) Tyrosine decomposition negative (28) Growth factorrequest none (29) Use of various carbon compounds Acetic acid + Adipicacid − Capric acid + Citric acid + Citraconic acid + Glycolic acid +Levulinic acid + Maleic acid + Malonic acid + Mesaconic acid + Muconicacid + Phenylacetic acid + Saccharic acid + Sebacic acid + D-Tartaricacid + m-Tartaric acid + L-Arabinose − Cellobiose − Fructose + D-Fucose− Glucose − Mannose − Maltose − Ribose − Rhamnose − Xylose − Mannitol −Gluconic acid − 2-Ketogluconic acid + N-Acetylglucosamine − Tryptamine −Ethanolamine − D-Alanine + L-Ornithine − L-Serine − L-Threonine −Glutamic acid + Benzoic acid + m-Hydroxybenzoic acid + Sodium salicinate− 2,3-Butylene glycol −

When these results were taxonomically examined based on Bergey's Manualof Systematic Bacteriology (1986), it was found that this strain belongsto the genus Pseudomonas but the properties thereof did not coincidewith those of standard strains. Therefore, this strain was namedPseudomonas sp. SD810.

Pseudomonas sp. SD811 Morphology: (1) Shape and size of cell rod 0.7-0.9μm × 1.5-3.0 μm (2) Motility motile (3) Gram staining negative (4) Sporenone (5) Bacteriolysis by 3% KOH positive Physiological activity: (1)Aminopeptidase positive (2) Oxidase positive (3) Catalase positive (4)Production of indole negative (5) Vp test negative (6) Reduction ofnitrate negative (7) Denitrification negative (8) Use of citric acid(Simons) positive (9) Urease negative (10) Phenylalanine deaminasenegative (11) Use of malonic acid positive (12) Production of levan fromsucrose negative (13) Lecithinase negative (14) Hydrolysis of starchnegative (15) Hydrolysis of gelatin negative (16) Hydrolysis of caseinnegative (17) Hydrolysis of DNA negative (18) Hydrolysis of Tween 80positive (19) Hydrolysis of exrin negative (20) Growth Behavior tooxygen obligately aerobic Growth at 37° C. − Growth at 41° C. − Growthat pH 5.6 + Growth in Mac-Conkey-Agar medium − Growth in SS-Agar medium− Growth in Cetrimid-Agar − (21) Production of dye Nondiffusive negativeDiffusive negative Fluorescent negative Pyrocyanine negative (22) OFTest no decomposition of sugar (23) Formation of acids Glucose positiveFructose positive Xylose positive (24) ONPG (β-galactosidase) negative(25) Arginine dihydrolase negative (26) Production of gas from glucosenegative (27) Tyrosine decomposition negative (28) Growth factor requestnone (29) Use of various carbon compounds Acetic acid + Adipic acid −Capric acid − Citric acid − Citraconic acid + Glycolic acid + Levulinicacid − Maleic acid + Malonic acid + Mesaconic acid − Muconic acid +Phenylacetic acid + Saccharic acid + Sebacic acid − D-Tartaric acid +m-Tartaric acid − L-Arabinose + Cellobiose − Fructose + D-Fucose +Glucose + Mannose − Maltose − Ribose + Rhamnose + Xylose − Mannitol +Gluconic acid − 2-Ketogluconic acid − N-Acetylglucosamine + Tryptamine −Ethanolamine − D-Alanine − L-Ornithine − L-Serine − L-Threonine −Glutamic acid + Benzoic acid + m-hydroxybenzoic acid + Sodium salicinate− 2,3-Butylene glycol −

When these results were taxonomically examined in the same manner basedon Bergey's Manual of Systematic Bacteriology, it was found that thisstrain belongs to the genus Pseudomonas but the properties thereof didnot coincide with those of standard strains. Therefore, this strain wasnamed Pseudomonas sp. SD811.

Pseudomonas sp. SD812 Morphology: (1) Shape and size of cell rod 0.5-0.8μm × 1.5-3.0 μm (2) Motility motile (3) Gram staining negative (4) Sporenone (5) Bacteriolysis by 3% KOH positive Physiological activity: (1)Aminopeptidase positive (2) Oxidase positive (3) Catalase positive (4)Production of indole negative (5) VP Test negative (6) Reduction ofnitrate negative (7) Denitrification negative (8) Use of citric acid(Simons) positive (9) Urease positive (10) Phenylalanine deaminasenegative (11) Use of malonic acid positive (12) Production of levan fromsucrose negative (13) Lecithinase negative (14) Hydrolysis of starchnegative (15) Hydrolysis of gelatin negative (16) Hydrolysis of caseinnegative (17) Hydrolysis of DNA negative (18) Hydrolysis of Tween 80negative (19) Hydrolysis of exrin negative (20) Growth Behavior tooxygen obligately aerobic Growth at 37° C. − Growth at 41° C. − Growthat pH 5.6 + Growth in Mac-Conkey-Agar medium − Growth in SS-Agar medium− Growth in Cetrimid-Agar − (21) Production of dye Nondiffusive positiveDiffusive negative Fluorescent negative Pyrocyanine negative (22) OFTest no decomposition of sugar (23) Formation of acids Glucose negativeFructose negative Xylose negative (24) ONPG β-galactosidase) negative(25) Arginine dihydrolase negative (26) Production of gas from glucosenegative (27) Tyrosine decomposition positive (28) Growth factor requestnone (29) Use of various carbon compounds Acetic acid + Adipic acid +Capric acid − Citric acid + Citraconic acid − Glycolic acid + Levulinicacid − Maleic acid + Malonic acid + Mesaconic acid + Muconic acid +Phenylacetic acid + Saccharic acid + Sebacic acid + D-Tartaric acid −m-Tartaric acid + L-Arabinose − Cellobiose − Fructose − D-Fucose −Glucose − Mannose − Maltose − Ribose − Rhamnose − Xylose − Mannitol −Gluconic acid + 2-Ketogluconic acid − N-Acetylglucosamine − Tryptamine −Ethanolamine − D-Alanine + L-Ornithine − L-Serine − L-Threonine +Glutamic acid + Benzoic acid + m-hydroxybenzoic acid + Sodium salicinate− 2,3-Butylene glycol −

When these results were taxonomically examined in the same manner basedon Bergey's Manual of Systematic Bacteriology, it was found that thisstrain belongs to the genus Pseudomonas but the properties thereof didnot coincide with those of standard strains. Therefore, this strain wasnamed Pseudomonas sp. SD812.

Burkholderia sp. SD816 Morphology: (1) Shape and size of cell rod (2)Motility motile (3) Gram staining negative (4) Spore none (5) FlagellaFlagallation state is unknown. Physiological activity: (1) Oxidasepositive (2) Catalase positive (3) Cleavage of protocatechinic acidortho type (4) Reduction of nitrate negative (5) Denitrificationnegative (6) Accumulation of PHB positive (7) Hydrolysis of starchnegative (8) Hydrolysis of gelatin negative (9) Growth Behavior tooxygen obligately aerobic Growth at 40° C. + (10) Production of dye Hueof colony no production of characteristic colonial dye Production ofwater-soluble dye negative (11) OF Test no decomposition of sugar (12)Arginine dihydrolase negative (13) Use of various carbon compoundsLevulinic acid − Mesaconic acid − D-Tartaric acid + Ribose + Rhamnose +Xylose + Tryptamine − 2,3-Butylene glycol − (14) Quinone type Q-8 (15)GC content of DNA in cell (mol %) 62

When these results were taxonomically examined in the same manner basedon Bergey's Manual of Systematic Bacteriology (1986, 1994), it was foundthat this strain belongs to the genus Burkholderia but the propertiesthereof did not coincide with those of standard strains. Therefore, thisstrain was named Burkholderia sp. SD816.

α-Halocarbonyl compounds having an α,β-carbon-carbon double bond, whichcan be suitably used in the present invention, is represented by thefollowing formula (1):

wherein R₁ represents a halogen atom, preferably a chlorine atom or abromine atom; R₂ and R₃ each independently represents a hydrogen atom, ahalogen atom, a linear or branched aliphatic hydrocarbon group havingfrom 1 to 6 carbon atoms, a linear or branched alkoxy group having from1 to 6 carbon atoms, a hydroxyl group, a carboxyl group, an aromaticgroup which may be substituted, or a saturated or unsaturated nitrogen-,oxygen- or sulfur-containing heterocyclic group, preferably a hydrogenatom; R₄ represents a hydroxyl group, a linear or branched alkoxy grouphaving from 1 to 4 carbon atoms, or a primary, secondary or tertiaryamino group, preferably a hydroxyl group.

Specific examples of the compounds include α-chloroacrylic acid,α-bromoacrylic acid, 2-chloro-2-butenoic acid, 2-bromo-2-butenoic acid,2-chloro-2-pentenoic acid, 2-bromo-2-pentenoic acid, and the methylester and the ethyl ester thereof. Among these, α-chloroacrylic acid andα-bromoacrylic acid are preferred.

In the present invention, the reaction is performed by contacting amicroorganism belonging to the genus Pseudomonas or Burkholderia,specifically a microorganism such as Pseudomonas sp. SD811 orBurkholderia sp. SD816 strain, with an α-halocarbonyl compound having anα,β-carbon-carbon double bond to reduce the carbon—carbon double bond,thereby producing a corresponding α-halo-α,β-saturated carbonylcompound.

For the reduction of the carbon—carbon double bond of an α-halocarbonylcompound having an α,β-carbon-carbon double bond performed in thepresent invention, a microbial cell obtained by the cultivationaccording to the above-described method or a microbial product of themicroorganism, such as a cell-free extract obtained by disrupting a cellculture according to the above-described method, may be used underconditions such that the reducing activity of the microorganism can bestably achieved.

More specifically, in the case of using a cell obtained by cultivation,an α-halocarbonyl compound having an α,β-carbon-carbon double bond iscontinuously or batchwise added to a culture suspension as a substrateto a concentration of from 0.1 to 10%, preferably from 0.2 to 2%, andcultivation is performed at a growth temperature of from 15 to 35° C.,preferably from 25 to 30° C., thereby producing a correspondingα,β-saturated carbonyl compound in the culture suspension.

Alternatively, the culture obtained by the above-described method issubjected to centrifugation or the like to recover cells and the cellsare suspended in an appropriate solution, for example, an aqueoussolution such as a diluted pH buffer solution. To this suspension, anα-halocarbonyl compound having an α,β-carbon-carbon double bond iscontinuously or batchwise added as a substrate to a concentration, forexample, of from 0.1 to 10% and reacted at a temperature of from 15 to50° C., preferably from 25 to 30° C., while adjusting the reaction pH tofrom 6.0 to 9.0, preferably from 6.5 to 7.3, thereby producing acorresponding α-halo-α,β-saturated carbonyl compound in the cellsuspension.

In the case of using a microbial product of a microorganism, forexample, the culture obtained by the above-described culture method issubjected to centrifugation to recover cells, the cells are disrupted byFrench pressing or a like method to obtain a cell-free extract, and thecell-free extract is added to a reaction mixture containing anα-halocarbonyl compound having an α,β-carbon-carbon double bond as asubstrate, in a concentration of from 0.1 to 10%, preferably from 0.2 to2%, and also containing an ingredient effective in maintaining the pH ofthe reaction mixture, in a concentration of from 10 mM to 1 M, andreacted with continuous or batch addition of substrate at a temperatureof from 15 to 50° C., preferably from 28 to 35° C., thereby producing acorresponding α,β-saturated carbonyl compound.

In the present invention, the reaction may be performed whilecontinuously or batchwise adding a substance effective in maintainingthe activity of reducing the α-halocarbonyl compound having anα,β-carbon-carbon double bond, for example, a compound capable of beingoxidized by the microorganism used, such as sugar or organic acid,preferably glucose or L-lactic acid, by itself or as a mixed solutionwith an α-halocarbonyl compound having an α,β-carbon-carbon double bondto have a concentration of from 0.1 to 10%, preferably from 0.2 to 1%during the reaction. The ratio of the α-halocarbonyl compound having anα,β-carbon-carbon double bond to the additional substance to be oxidizedmay be freely selected between 1:1 and 20:1 on a molar basis. By thisaddition of sugar or organic acid, the reaction time may be prolongedand in turn, the concentration of the objective productα-halo-α,β-saturated carbonyl compound in the reaction suspension may beincreased. This is advantageous for collecting the product by isolation.Except for the cultivation time, the reaction may be performed either inan aerobic or anaerobic environment. The ratio of the cell or cell-freeextract to the α-halocarbonyl compound having an α,β-carbon-carbondouble bond as the substrate, and the timing and rate or frequency ofaddition may be freely selected in the range capable of attaining thecompletion of reaction within the objective time.

In the present invention, the α-halo-α,β-saturated carbonyl compoundobtained by the reduction of an α-halocarbonyl compound having anα,β-carbon-carbon double bond is a metabolic intermediate for themicroorganism used and may be further decomposed. For example, somemicroorganisms relatively swiftly decompose the α-chloropropionic acidproduced from α-chloroacrylic acid. If the case is so, the decompositionreaction may be stopped by using a mutant defective in decomposingactivity, decreasing the pH value, heat-treating the cells or cell-freeextract, or adding an appropriate inhibitor of the decomposing enzyme.To speak more specifically, under the conditions where anα-chloropropionic acid is produced from an α-chloroacrylic acid at anoptimal rate, the Pseudomonas sp. SD811 strain usually swiftlydecomposes the α-chloropropionic acid produced in the culture suspensionor reaction suspension, whereby the conversion ratio intoα-chloropropionic acid based on the α-chloroacrylic acid decreases.However, since the optimal reaction pH at the stage of producingα-chloropropionic acid from α-chloroacrylic acid is from 5 to 7 and theoptimal reaction pH at the decomposition stage of α-chloropropionic acidis from 7.0 to 7.3, thus, the optimal reaction pH at the decompositionstage of α-chloropropionic acid is higher than the optimal reaction pHat the stage of producing α-chloropropionic acid from α-chloroacrylicacid. Accordingly, the amount of α-chloropropionic acid produced can beincreased by performing the reaction at a low pH of from 5 to 7 which isthe optimal reaction pH range for the stage of producingα-chloropropionic acid. Furthermore, certain enzymes which decompose theα-chloropropionic acid are known to be effectively inhibited byhydroxylamine (see, Soda K. et al., J. Biol. Chem., 272, 3363-3368(1997)) and also in the present invention, the decomposition reactioncan be inhibited by adding an appropriate amount of hydroxylamine to theculture suspension or reaction suspension.

The cell or cell-free extract of the microorganism for use in thepresent invention may be used by fixing it to an immobilizing support ofvarious types by a commonly known method such as adsorption, inclusionor crosslinking. The kind of the support is not particularly limited andfor example, a polysaccharide-type material such as cellulose, apolymer-type material, or a protein-type material such as collagen, maybe used.

The α-halocarbonyl compound having an α,β-carbon-carbon double bond foruse in the present invention is a molecule prochiral at the α-position,however, by reducing the carbon—carbon double bond, a correspondingα-halo-α,β-saturated carbonyl compound having an absolute S formconfiguration at the α-position may be produced.

The culture method of the microorganism used in the present invention isnot particularly limited as long as aerobic microorganisms in generalcan grow. The carbon source of the medium may be any source as long asthe above-described microorganisms can be used and examples thereofinclude saccharides, acetic acid, lactic acid and a mixture thereof.Examples of nitrogen sources which can be used include ammonium saltssuch as ammonium sulfate and ammonium phosphate, nitrogen-containingcompounds such as meat extract and yeast extract, and mixtures thereof.

In addition to these ingredients, nutrients commonly used incultivation, such as inorganic salts, trace metal salts and vitamins,may be used in the medium by appropriately mixing them. Furthermore, ifdesired, a factor of accelerating the growth of the microorganism and aningredient effective in maintaining the pH of the medium may be added.Also, a compound effective in increasing the reductive activity of themicroorganism, for example, an α-halocarbonyl compound having anα,β-carbon-carbon double bond, such as α-chloroacrylic acid, may be usedas a sole carbon source or may be used by mixing it with a plurality ofcarbon sources.

The microorganism for use in the present invention may be cultured underaerobic conditions used for the cultivation of almost all aerobes orfacultative anaerobes, for example, under conditions such that the pH ofthe medium is from 5.5 to 8.0, preferably from 6.5 to 7.0, and thegrowth temperature is from 15 to 35° C., preferably from 25 to 30° C.The cultivation time is, for example, from 1 to 144 hours, preferablyfrom 12 to 72 hours.

The α-halo-α,β-saturated carbonyl compound produced according to thepresent invention may be obtained using an ordinary purification methodsuch as organic solvent extraction or distillation. For example,α-chloropropionic acid produced from α-chloroacrylic acid may beobtained by subjecting the culture suspension or reaction suspension toorganic solvent extraction, distillation or the like. Furthermore,although the α-halocarbonyl compound having an α,β-carbon-carbon doublebond is a molecule prochiral at the α-position, the α-halo-α,β-saturatedcarbonyl compound produced by the reducing method of the presentinvention is a chiral compound and it is advantageous to determine thepurity of the enantiomer thereof by GC and/or HPLC with a chiral columnor by a polarimeter.

As described in the foregoing, in the method of the present inventionfor producing a corresponding α-halo-α,β-saturated carbonyl compoundhaving an absolute S form configuration from an α-halocarbonyl compoundhaving an α,β-carbon-carbon double bond by reducing the carbon—carbondouble bond, an aerobe or facultative anaerobe is used, therefore, themethod is favored with high profitability, good operability andexcellent processing safety.

The present invention is described in greater detail below by referringto the Examples, however, the present invention should not be construedas being limited to these Examples.

EXAMPLE 1

Detection of Activity of Reducing α-Halocarbonyl Compound Having anα,β-Carbon-Carbon Double Bond

Microorganisms cultured by accumulative cultivation or accumulativeacclimatization and isolated were cultured at 30° C. using a mediumobtained by adding 0.2% of α-chloroacrylic aid or α-chloro-α,β-butenoicacid as a carbon source to a basal medium containing yeast extract (0.5g/l), ammonium sulfate (2 g/l), sodium dihydrogenphosphate (1 g/l),dipotassium hyrogenphosphate (1 g/l) and magnesium sulfate (0.1 g/l)(for the isolation of some microorganisms which grow only in a high saltconcentration medium, a medium obtained by adding sodium chloride in aconcentration of from 10 to 20% to the above-descried basal medium wasused as the basal medium). Whether or not α-chloropropionic acid orα-chlorobutyric acid as the corresponding reduction product appeared inthe culture suspension was examined using gas chromatography. From eachsolution, 0.5 ml was sampled at a specific time and centrifuged toremove cells. Thereafter, 0.4 ml of the supernatant was mixed with 0.4ml of 2N HCl and analyzed under the following conditions:

Apparatus: GC-7A (manufactured by Shimadzu Seisakusho) Column:Thermon-3000/SHINCARBON A, 2.6 mm × 2.1 m Carrier gas: nitrogen, 50ml/mm. Detection: FID Column temperature: 200° C. (constant) Injection:2 to 10 μl, 260° C. Recording: CHROMATOCODER 12 (SIC)

By this detection, peaks appeared swiftly after the initiation ofcultivation at the position of α-chloropropionic acid or α-chlorobutyricacid accompanying the consumption of α-chloroacrylic in the culturesuspension of a plurality of microorganisms. The peaks were analyzed byGC-MS and found to coincide with the mass spectrum of α-chloropropionicacid or α-chlorobutyric acid as the standard substance, therefore, theproducts were confirmed to be α-chloropropionic acid or α-chlorobutyricacid. The genera of microorganisms of which cultivation brought aboutgeneration of the reduction product were identified, as a result, it wasfound that microorganisms belonging to various genera have reducingactivity. Microorganisms recognized to have the activity were aerobes orfacultative anaerobes belonging to the genera Acetobacter, Actinomyces,Acinetobacter, Agrobacterium, Aeromonas, Alcaligenes, Arthrobacter,Azotobacter, Bacillus, Brevibacterium, Burkholderia, Cellulomonas,Corynebacterium, Enterobacter, Enterococcus, Escherichia,Flavobacterium, Gluconobacter, Halobacteium, Halococccus, Klebsiella,Lactobacillus, Microbacterium, Micrococcus, Micropolyspora,Mycobacterium, Nocardia, Pseudomonas, Pseudonocardia, Rhodococcus,Rhodobacter, Serratia, Staphylococcus, Streptococcus, Streptomyces andXanthomonas. Strains of the genus Pseudomonas prevailed in the strainsrecognized. Microorganisms relatively surpassed in the amount of thereduction product were the microorganisms belonging to the generaPseudomonas and Burkholderia. With any strain, the reduction productrecognized in the culture suspension was in a small amount (0.01% orless), therefore, the following elite strains were examined foroptimization of product accumulating conditions.

EXAMPLE 2

(1) Cultivation of Pseudomonas sp. SD811 Strain

Pseudomonas sp. SD811 strain was cultured in a medium containing thefollowing ingredients (amount: g/L): α-chloroacrylic acid (2), yeastextract (0.5), ammonium sulfate (2), sodium dihydrogenphosphate (1),dipotassium hydrogenphosphate (1) and magnesium sulfate (0.1). Themedium was prepared as follows. All ingredients except forα-chloroacrylic acid and magnesium sulfate were dissolved in 950 ml ofwater and after the pH was adjusted to 7.0, the solution was poured intoa 5 l-volume flask and sterilized at 121° C. for 20 minutes. After thetemperature of this medium decreased to about 70° C., a solutionobtained by dissolving α-chloroacrylic acid and magnesium sulfate in 50ml of water, adjusted to a pH of 7.0 and then sterilized through asterilization filter was mixed with the medium prepared above. Withoutsupplying oxygen or adjusting the pH any more, a 5% seed culture (OD 660nm: 1.10) was inoculated in this medium and the strain was cultured at30° C.

(2) Detection of α-Chloropropionic Acid in α-Chloroacrylic Acid CultureMedium

During cultivation of the Pseudomonas sp. SD811 strain withα-chloroacrylic acid, 0.5 ml was sampled at a specific time. The samplewas centrifuged to remove cells and 0.4 ml of the supernatant was mixedwith 0.4 ml of 2N HCl. The solution obtained was analyzed under theconditions described in Example 1.

By the detection, a peak appeared swiftly after the initiation ofculturing at the position of α-chloropropionic acid accompanying theconsumption of α-chloroacrylic acid in the culture suspension ofPseudomonas sp. SD811 strain. The production was about 0.02% of theculture suspension at the maximum time and the conversion ratio based onthe substrate α-chloroacrylic acid was about 10%.

EXAMPLE 3

(1) Cell Suspension Reaction 1 Using α-Chloroacrylic Acid as Substrate

A culture obtained by culturing the Pseudomonas sp. SD811 strain in a{fraction (1/10)} scale of the method in Example 2 was subjected tocentrifugation to recover the cells. The cells were suspended in 20 mlof a solution (adjusted to a pH of 7.3) containing 0.2% ofα-chloroacrylic acid and 100 mM of phosphate buffer (pH: 7.3) andreacted by shaking at 28° C.

From the reaction mixture, 0.5 ml was sampled at a specific time andcentrifuged to remove cells. Thereafter, 0.4 ml of the supernatant and0.1 ml of 6N HCl were mixed and then the product was extracted with 0.4ml of ethyl acetate. The sample extracted was analyzed by the method ofExample 1. As a result, a peak appeared at the position ofα-chloropropionic acid accompanying the consumption of α-chloroacrylicacid in the reaction mixture. The production was about 0.05% of thereaction suspension at the maximum time and the conversion ratio wasabout 25% based on the substrate α-chloroacrylic acid.

(2) Isolation of α-Chloropropionic Acid from Cell Suspension ReactionUsing α-Chloroacrylic Acid as Substrate

A culture obtained by culturing the Pseudomonas sp. SD811 strain in a ⅕scale of the method in Example 1 was subjected to centrifugation torecover the cells and the cells were suspended in 100 ml of a solution(adjusted to a pH of 7.3) containing 0.2% of α-chloroacrylic acid and100 mM of potassium phosphate-sodium hydroxide buffer and reacted bystirring at 28° C. At the time when the α-chloroacrylic acid wasexhausted during the reaction, α-chloroacrylic acid was added in aconcentration of 0.2% of the reaction mixture and the reaction wascontinued. From the reaction suspension, 0.5 ml was sampled at aspecific time, the product was separated by extraction according to themethod of Example 3, and the production of α-chloropropionic acid wasmonitored by the method of Example 1. After about 9 hours whenα-chloroacrylic acid was not detected in the reaction suspension, thereaction was completed and the entire amount of the reaction suspensionwas subjected to centrifugation to remove cells. To 95 ml of thesupernatant obtained, 20 ml of 6N HCl was added, and the product wasextracted with 95 ml of ethyl acetate. The ethyl acetate layer waswashed with 100 ml of saturated saline and then concentrated by removingethyl acetate by evaporation. The concentrated sample was analyzed bythe method of Example 1, as a result, a slight amount of α-chloroacrylicacid and α-chloropropionic acid were detected. The α-chloropropionicacid present in the concentrated sample was about 100 mg in total andthe conversion ratio was about 25% based on the substrateα-chloroacrylic acid.

EXAMPLE 4

(1) Methylation of α-Chloropropionic Acid

When optical resolution is performed by an optical resolution GC column,unmodified carboxylic acid exhibits poor separability due to the effectof the carboxyl group in many cases. Therefore, the product was methylesterified by a boron trifluoride-methanol complex salt method. Morespecifically, 4 ml of methanol was added to 3 mg of α-chloropropionicacid and mixed. Thereafter, 1 ml of a 14% methanol solution of borontrifluoride-methanol complex was further added and the resultingsolution was refluxed on an oil bath at 80° C. for 1 hour whilestirring. After 1 hour, 30 ml of water was added to the reaction mixtureand the reactant was extracted with 10 ml of ethyl acetate. The ethylacetate was then distilled off by a centrifugal evaporator toconcentrate the sample to the entire amount of about 0.1 ml. Theconcentrated sample was analyzed by the method of Example 1 except thatthe column temperature only was changed to 120° C. As a result, one mainpeak was observed at the position of methyl α-chloropropionate. By theGC-MS analysis of the peak, the product was identified to be methylα-chloropropionate.

(2) Optical Resolution GC Analysis of α-Chloropropionic Acid

After the methylation by the above-described method, the S-formα-chloropropionic acid and the R-form α-chloropropionic acid could besuccessfully separated under the following analysis conditions.

Apparatus: GC-14A (manufactured by Shimadzu Seisakusho) Column:CP-Chirasil-DEX CB, 0.32 mm I.D. × 25 m, df = 0.25 mm (manufactured byGL Science K.K.) Carrier gas: He, 0.38 kg/cm² Detection: FID, 275° C.Column temperature: 70° C. (constant) Injection: 0.1 to 0.2 μl, split,about 1:100, 250° C. Recording: CHROMATOPACK C-6A (manufactured byShimadzu Seisakusho)

The methyl α-chloropropionate obtained from the reaction mixture ofExample 3 under the above-described conditions was analyzed, then a peakwas observed only at the position of S-form methyl α-chloropropionate.The optical purity thereof was 99% or more.

EXAMPLE 5

Cell Suspension Reaction 2 Using α-Chloroacrylic Acid as Substrate

A culture obtained by culturing Pseudomonas sp. SD811 strain in a{fraction (1/10)} scale of the method as described in Example 2 wassubjected to centrifugation to recover the cells. The cells weresuspended in 20 ml of a solution (adjusted to a pH of 5.7) containing0.2% of α-chloroacrylic acid and 100 mM of phosphate buffer (pH: 5.7)and reacted by shaking at 28° C. From the reaction mixture, 0.5 ml wassampled at a specific time and centrifuged to remove cells. Thereafter,0.4 ml of the supernatant and 0.1 ml of 6N HCl were mixed and then theproduct was extracted with 0.4 ml of ethyl acetate. The sample extractedwas analyzed by the method of Example 1, as a result, a peak appeared atthe position of α-chloropropionic acid accompanying the consumption ofα-chloroacrylic acid in the reaction mixture. The production was about0.08% of the reaction suspension at the maximum time and the conversionratio was about 41% based on the substrate α-chloroacrylic acid.

EXAMPLE 6

Preparation of α-Chloropropionic Acid-Undecomposable Mutant

A culture obtained by culturing Pseudomonas sp. SD811 strain in a{fraction (1/200)} scale of the method in Example 2 was subjected tocentrifugation to recover the cells and the cells were washed withphysiological saline. The washed cells were re-suspended in 1 ml of 10to 100 mM phosphate buffer (pH: 7). To the resulting cell suspension,N-methyl-N′-nitro-N-nitrosoguanidine (NTG) was added to a finalconcentration of from 50 to 100 ppm and the cells were treated at roomtemperature for from 2 to 20 minutes. After the treatment, the cellsuspension was subjected to centrifugation to recover the cells, thecells were washed with sterilized physiological saline, and the entireamount thereof was inoculated in 5 ml of an L medium (polypeptone: 10g/l, yeast extract: 5 g/l, sodium chloride: 5 g/l, pH: 7) and culturedby shaking at 28° C. After the completion of cultivation, the culturewas subjected to centrifugation to recover the cells and the cells weresuspended in a 20% glycerin solution. The suspension was equally dividedby an appropriate amount and frozen to prepare glycerin stocks forselection of a mutant.

EXAMPLE 7

Selection of α-Chloropropionic Acid-Undecomposanble Mutant

A glycerin stock for selection of a mutant prepared above was inoculatedin 5 ml of a medium used in the method of Example 2 and cultured at 28°C. for 5 hours. At the time when the turbidity was increased as high asseveral times, penicillin G was added to the culture suspension in anamount of giving a final concentration of from 100 to 1,000 ppm and thecultivation was continued at 28° C. After the cultivation for from 5 to16 hours, the culture suspension was subjected to centrifugation torecover the cells. The cells were washed twice with sterilizedphysiological saline and the entire amount thereof was inoculated in 5ml of L-broth and cultured by shaking at 28° C. an entire day and night.

The culture suspension obtained above was diluted, spread on a mediumthe same as used in Example 2 except that the α-chloroacrylic acid as acarbon source was replaced by an equivalent amount of lactic acid andthe medium was solidified by adding 2% agar, and cultured at 28° C.After 1 or 2 days, colonies formed on the plate were replicated on themedium used as described in Example 2 which was solidified by adding 2%agar, and cultured at 28° C. for 1 or 2 days. Strains which grew on thelactic acid plate but did not grow on the α-chloroacrylic acid platewere isolated as candidates for the α-chloropropionicacid-undecomposable mutant.

The candidate strains each isolated were inoculated by an inoculatingloop in 5 ml of a medium the same as used in Example 2 except that 2 g/lof lactic acid was added as a carbon source capable of growing, andcultured by shaking at 28° C. The culture suspensions obtained wereanalyzed by the method of Example 1, as a result, the production maximumof α-chloropropionic acid produced in the culture was distributeddepending on the candidate strains, however, the production maximum withthe mutant of Pseudomonas sp. SD811 was about 0.17% of the culturesuspension and the conversion ratio was about 85% based on the substrateα-chloroacrylic acid. With the variation strain of Burkholderia sp.SD816, the production maximum was about 0.15% of the culture suspensionand the conversion ratio was about 75% based on the substrateα-chloroacrylic acid. These strains were selected as theα-chloropropionic acid-undecomposable mutant.

EXAMPLE 8

Cell Suspension Reaction 3 Using α-Chloroacrylic Acid as Substrate

The α-chloropropionic acid-undecomposable mutant of Pseudomonas sp.SD811 and the α-chloropropionic acid-undecomposable mutant ofBurkholderia sp. SD816 each was cultured in the same medium of lacticacid as used in Example 7, and each of the cultures obtained wassubjected to centrifugation to recover the cells. The cells of eachstrain were suspended in 20 ml of a solution (adjusted to a pH of 7.3)containing 0.2% of α-chloroacrylic acid and 100 mM of phosphate buffer(pH: 7.3) and reacted by shaking at 28° C.

From each reaction suspension, 0.5 ml was sampled at a specific time andcentrifuged to remove cells. To 0.4 ml of the supernatant, 0.1 ml of 6NHCl was mixed and then, the product was extracted with 0.4 ml of ethylacetate. The sample extracted was analyzed by the method of Example 1.As a result, a peak appeared at the position of α-chloropropionic acidaccompanying the consumption of α-chloroacrylic acid in the reactionsuspension. With the mutant of Pseudomonas sp. SD811, the production wasabout 0.19% of the reaction suspension at the time when theα-chloroacrylic acid disappeared in the reaction mixture and theconversion ratio was about 95% based on the substrate α-chloroacrylicacid. With the mutant of Burkholderia sp. SD816, those were about 0.2%and about 100%, respectively. In either case, the α-chloropropionic acidproduced did not decrease with the passing of time. The optical activityof the α-chloropropionic acid was analyzed according to the method ofExample 4. As a result, in both cases, the product was an S-formcompound and the optical purity thereof was 99% or more.

EXAMPLE 9

Cell Suspension Reaction 4 Using α-Chloroacrylic Acid as Substrate

The α-chloropropionic acid-undecomposable mutant of Pseudomonas sp.SD811 and the α-chloropropionic acid-undecomposable mutant ofBurkholderia sp. SD816 each was cultured in 100 ml of a medium obtainedby adding 0.2% of glucose as a carbon source to the basal medium used inExample 2, and the cultures obtained each was subjected tocentrifugation to recover the cells. The cells of each strain weresuspended in 20 ml of a solution (adjusted to a pH of 7.3) containing0.2% of α-chloroacrylic acid and 100 mM of phosphate buffer (pH: 7.3)and reacted by shaking at 28° C.

From each reaction suspension, 0.5 ml was sampled at a specific time andcentrifuged to remove cells. To 0.4 ml of the supernatant, 0.1 ml of 6NHCl was mixed and then, the product was extracted with 0.4 ml of ethylacetate. The sample extracted was analyzed by the method of Example 1.As a result, after the lag time of a few hours at the initial stage whenthe reaction was started, a peak appeared at the position ofα-chloropropionic acid accompanying the consumption of α-chloroacrylicacid in the reaction suspension. With the mutant of Pseudomonas sp.SD811, the production was about 0.19% of the reaction suspension at thetime when the α-chloroacrylic acid disappeared in the reaction mixtureand the conversion ratio was about 95% based on the substrateα-chloroacrylic acid. With the mutant of Burkholderia sp. SD816, thosewere about 0.2% and about 100%, respectively.

EXAMPLE 10

Cultivation in Jar Fermenter

The α-chloropropionic acid-undecomposable mutant of Burkholderia sp.SD816 was cultured for 16 hours in 100 ml of a medium (5×medium, pH:7.0) the same as used in Example 9 except that the medium ingredientseach had a five-fold concentration. The culture obtained was inoculatedin 2 L of 5×medium filled in a 5 L-volume jar fermenter and cultured at28° C., 800 rpm and an aeration rate of 1 ml/min. When from about 15 to20 hours passed after the initiation of cultivation, the glucoseinitially charged were completely consumed, accordingly, a 5 to 20%glucose solution, a 5 to 15% ammonium sulfate solution and a 1 to 5%yeast extract solution as individual solutions or a mixed solution werefurther continuously added by means of PERISTACK pump. The addition ratewas controlled so that the glucose concentration of from 0.02 to 2%could be maintained during the addition. The pH of the culturesuspension was adjusted by a 20% aqueous ammonia to lie in the range offrom 6.3 to 7.3. After the cultivation for from about 30 to 48 hours bythis method, OD 660 nm was about 30 to 50.

EXAMPLE 11

Cell Suspension Reaction 5 Using α-Chloroacrylic Acid as Substrate

The α-chloropropionic acid-undecomposable mutant of Burkholderia sp.SD816 was cultured according to the method of Example 10. When from 48to 72 hours passed after the initiation of cultivation, the addition ofglucose and the like was stopped to interrupt growth. To this culturesuspension, α-chloroacrylic acid was added to a final concentration ofabout 0.2% and reacted under the same conditions as in the cultivation.From the reaction suspension, 0.5 ml was sampled at a specific time andcentrifuged to remove cells. Thereafter, 0.4 ml of the supernatant and0.4 ml of 2N HCl were mixed and the α-chloroacrylic acid concentrationin the solution was determined under the following conditions.

Apparatus: LC-9A (manufactured by Shimadzu Seisakusho) Column: ODSpakF-511/4.6 mm × 250 mm (Shodex) Eluent acetonitrile/water = 2/8 + 0.1%trifluoroacetic acid, 1 ml/min. Detection: SPD-6AV UV-VISSpectrophotometer (manufactured by Shimadzu Seisakusho) Columntemperature: 25° C. Injection: Autosampler Model 23 (SIC) with 20 μlsample loop Recording: CHROMATOCODER 12 (SIC)

In this reaction, after the lag time of from 3 to 7 hours at the initialstage when the reaction was started, consumption of α-chloroacrylic acidstarted. When the α-chloroacrylic acid concentration became about 0.02%,α-chloroacrylic acid was further added so that the α-chloroacrylic acidconcentration could be increased to about 0.2%. This operation wasrepeated until the consumption of α-chloroacrylic acid substantiallyterminated. The accumulative production of α-chloropropionic acid after32 hours where the reaction was substantially stopped was from about 1.0to 1.2% of the reaction suspension and the conversion ratio was about97% based on the substrate α-chloroacrylic acid. The optical activity ofthe α-chloropropionic acid accumulated was analyzed according to themethod of Example 4. As a result, the product was an S-form compound andthe optical purity thereof was 99% or more.

EXAMPLE 12

Cell Suspension Reaction 6 Using α-Chloroacrylic Acid as Substrate

The α-chloropropionic acid-undecomposable mutant of Burkholderia sp.SD816 was cultured for from 48 to 72 hours according to the method ofExample 10 and the culture obtained was subjected to centrifugation torecover the cells. The cells were suspended in 2 l of a solution(adjusted to a pH of 7.1) containing 0.2% α-chloroacrylic acid and 60 mMof phosphate buffer (pH: 7.1) and reacted at 28° C., 800 rpm and anaeration rate of 1 ml/min. From the reaction suspension, 0.5 ml wassampled at a specific time and centrifuged to remove cells. Thereafter,0.4 ml of the supernatant and 0.4 ml of 2N HCl were mixed and theα-chloroacrylic acid concentration in the solution was determined by themethod described in Example 11.

In this reaction, after a lag time of from 3 to 7 hours at the initialstage when the reaction was started, consumption of α-chloroacrylic acidstarted. When the α-chloroacrylic acid concentration became about 0.02%,α-chloroacrylic acid was further added so that the α-chloroacrylic acidconcentration could be increased to about 0.2%. This operation wasrepeated until the consumption of α-chloroacrylic acid substantiallyterminated. The accumulative production of α-chloropropionic acid after28 hours where the reaction was substantially stopped was from about 0.8to 1.0% of the reaction suspension and the conversion ratio was about98% based on the substrate α-chloroacrylic acid.

EXAMPLE 13

Cell Suspension Reaction 7 Using α-Chloroacrylic Acid as Substrate

The α-chloropropionic acid-undecomposable mutant of Burkholderia sp.SD816 was cultured for from 48 to 72 hours according to the method ofExample 10 and the culture obtained was subjected to centrifugation torecover the cells. The cells were suspended in 2 l of 60 mM phosphatebuffer (pH: 7.1) and reacted at 28° C., 800 rpm and an aeration rate of1 ml/min while adding from 5 to 10% of an α-chloroacrylic acid solutionto the cell suspension little by little by means of PERISTACK pump. Fromthe reaction suspension, 0.5 ml was sampled at a specific time andcentrifuged to remove cells. Thereafter, 0.4 ml of the supernatant and0.4 ml of 2N HCl were mixed and the α-chloroacrylic acid concentrationin the solution was determined by the method described in Example 11.

In this reaction, after a lag time of from 5 to 10 hours at the initialstage when the reaction was started, consumption of α-chloroacrylic acidstarted. The addition rate of the α-chloroacrylic acid solution wascontrolled in accordance with the consuming rate so that theα-chloroacrylic acid concentration could be in the range of from about0.02 to 0.2%, preferably around 0.1%. This operation was continued untilthe consumption of α-chloroacrylic acid substantially terminated. Theaccumulative production of α-chloropropionic acid after 24 hours wherethe reaction was substantially stopped was from about 0.8 to 1.2% of thereaction suspension and the conversion ratio was about 95% based on thesubstrate α-chloroacrylic acid.

EXAMPLE 14

Cell Suspension Reaction 8 Using α-Chloroacrylic Acid as Substrate

The α-chloropropionic acid-undecomposable mutant of Burkholderia sp.SD816 was cultured for from 48 to 72 hours according to the method ofExample 10 and the culture obtained was subjected to centrifugation torecover the cells. The cells were suspended in 2 l of 60 mM phosphatebuffer (pH: 7.1) and reacted at 28° C., 800 rpm and an aeration rate of1 ml/min while adding from 5 to 10% of an α-chloroacrylic acid solutionto the cell suspension little by little by means of PERISTACK pump. Atthe same time, from 5 to 10% of a sodium lactate solution was addedalone or as a mixed solution with α-chloroacrylic acid little by littleby means of PERISTACK pump. From the reaction suspension, 0.5 ml wassampled at a specific time and centrifuged to remove cells. Thereafter,0.4 ml of the supernatant and 0.4 ml of 2N HCl were mixed and theα-chloroacrylic acid concentration in the solution was determined by themethod described in Example 11. The concentration of lactic acid wasdetermined using lactate dehydrogenase. The pH of the reaction systemwas adjusted to from 6.5 to 7.3 using 20% aqueous ammonia and 2N HCl.

In this reaction, after the lag time of from 5 to 10 hours at theinitial stage when the reaction was started, consumption ofα-chloroacrylic acid started. Lactic acid was swiftly consumedimmediately after the initiation of reaction. The addition rate of theα-chloroacrylic acid solution was controlled in accordance with theconsuming rate so that the α-chloroacrylic acid concentration could liein the range of from about 0.02 to 0.2%, preferably around 0.1%. At thesame time, the added amount of lactic acid was controlled to prevent theconcentration from exceeding 0.4%. This operation was continued forabout 60 hours. In this Example, the reduction clearly continued andproceeded at a constant rate even after about 60 hours because lacticacid was present together. The accumulative production ofα-chloropropionic acid after the 60-hour reaction was from about 2.8 to3.2% of the reaction suspension and the conversion ratio was about 99%or more based on the substrate α-chloroacrylic acid.

EXAMPLE 15

Cell Suspension Reaction 9 Using α-Chloroacrylic Acid as Substrate

The α-chloropropionic acid-undecomposable mutant of Burkholderia sp.SD816 was cultured for from 48 to 72 hours according to the method ofExample 10 and the culture obtained was subjected to centrifugation torecover the cells. The cells were suspended in 2 l of 60 mM phosphatebuffer (pH: 7.1) and reacted at 28° C., 800 rpm and an aeration rate of1 ml/min while adding a mixed aqueous solution containing 10% of anα-chloroacrylic acid and 10% of glucose to the cell suspension little bylittle by means of PERISTACK pump. From the reaction suspension, 0.5 mlwas sampled at a specific time and centrifuged to remove cells.Thereafter, 0.4 ml of the supernatant and 0.4 ml of 2N HCl were mixedand the α-chloroacrylic acid concentration in the solution wasdetermined by the method described in Example 11. In the determinationof the glucose concentration, the reaction supernatant as the originalsolution obtained after the centrifugation was used intact and subjectedto the measurement by a glucose analyzer. The pH of the reaction systemwas adjusted to from 6.5 to 7.3 using 20% aqueous ammonia and 2N HCl. Inthis reaction, after a lag time of from 6 to 12 hours at the initialstage when the reaction was started, consumption of α-chloroacrylic acidstarted. The glucose was swiftly consumed particularly right after theinitiation of reaction but after the reducing activity was induced, theglucose was consumed at a constant rate. The addition rate of the mixedsolution was controlled so that the α-chloroacrylic acid concentrationcould lie in the range of from about 0.02 to 0.2%, preferably around 1%,and at the same time, the glucose concentration could be prevented fromexceeding 0.4%. This operation was continued for about 60 hours. In thisExample, the reduction reaction clearly continued and proceeded at aconstant rate even after about 60 hours because glucose was presenttogether. The conversion ratio was about 99% or more based on theα-chloroacrylic acid after the 60-hour reaction.

EXAMPLE 16

Cell Suspension Reaction 10 Using α-Chloroacrylic Acid as Substrate

The α-chloropropionic acid-undecomposable mutant of Burkholderia sp.SD816 was cultured for from 48 to 72 hours in the ½ scale of the methodof Example 10 and the culture obtained was subjected to centrifugationto recover the cells. The cells were suspended in 1 l of a solution(adjusted to a pH of 7.1) containing 0.2% of α-chloroacrylic acid, 0.2%of glucose and 60 mM of phosphate buffer (pH: 7.1), and reacted at 28°C., 800 rpm and an aeration rate of 1 ml/min. At the end point in a lagtime of from 5 to 10 hours after the initiation of reaction, air for theaeration was changed to nitrogen gas and thereafter, the reaction wasperformed in an anaerobic environment. From the reaction suspension, 0.5ml was sampled at a specific time and centrifuged to remove cells. Then,0.4 ml of the supernatant and 0.4 ml of 2N HCl were mixed and theα-chloroacrylic acid concentration in the solution was determined by themethod described in Example 11. At the same time, the glucoseconcentration was determined by a glucose analyzer. The pH of thereaction system was adjusted to from 6.5 to 7.3 using 20% aqueousammonia and 2N HCl.

In this reaction, after a lag time of from 5 to 10 hours at the initialstage when the reaction was started, consumption of α-chloroacrylic acidstarted. When the α-chloroacrylic acid concentration became about 0.02%,α-chloroacrylic acid or glucose was further added so that theα-chloroacrylic acid concentration could be increased to 0.2% or theglucose concentration could be around 0.1%. This operation was continuedfor about 60 hours. In this Example, the reduction reaction clearlycontinued and proceeded at a constant rate even after about 60 hoursbecause a substance to be oxidized was present together. The consumptionof glucose showed a marked decrease immediately after the reactionsystem was changed from an aerobic environment to an anaerobicenvironment and the total consumption was reduced to about {fraction(1/10)} or less that in the reaction in an aerobic environment. Theaccumulative production of α-chloropropionic acid after the 60-hourreaction was from about 2.5 to 3.2% of the reaction suspension and theconversion ratio was about 99% or more based on the substrateα-chloroacrylic acid.

In the method of the present invention for producing a correspondingα-halo-α,β-saturated carbonyl compound from an α-halocarbonyl compoundhaving an α,β-carbon-carbon double bond by reducing the carbon—carbondouble bond, an aerobe or facultative anaerobe is used, therefore, themethod is favored with high profitability, good operability andexcellent processing safety. Furthermore, according to the method of thepresent invention, a high-purity α-halo-α,β-saturated carbonyl compounduseful as a chiral building block of medical and agricultural chemicalsand the like is produced.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

What is claimed is:
 1. A biologically pure culture of Pseudomonas sp.SD810 and mutants thereof having an activity of reducing theα,β-carbon-carbon double bond of an α-halocarbonyl compound having anα,β-carbon-carbon double bond.
 2. A biologically pure culture ofPseudomonas sp. SD811 and mutants thereof having an activity of reducingthe α,β-carbon-carbon double bond of an α-halocarbonyl compound havingan α,β-carbon-carbon double bond.
 3. A biologically pure culture ofPseudomonas sp. SD812 and mutants thereof having an activity of reducingthe α,β-carbon-carbon double bond of an α-halocarbonyl compound havingan α,β-carbon-carbon double bond.
 4. A biologically pure culture ofBurkholderia sp. SD816 and mutants thereof having an activity ofreducing the α,β-carbon-carbon double bond of an α-halocarbonyl compoundhaving an α,β-carbon-carbon double bond.
 5. A microbial productcontaining the microorganism or obtained from the microorganismdescribed in any one of claims 1 to 4 having the activity of reducingthe α,β carbon—carbon double bond of an α-halocarbonyl compound havingan α,β carbon—carbon double bond.
 6. The microbial product as claimed inclaim 5, which is a microbial culture, a microbial extract, a microbialcell suspension or a microbial cell fixed to a support.